Figure 3:

In the SDH reaction, the acccumulation of [NBT_dfz] in muscle fibers was measured every 15 s across a 10 min period as the change in OD at 570 nm. A. Pictomicrographs show a representative example of the change in OD in type I, IIa and IIx/IIb DIAm fibers at 0, 5, and 10 min during the SDH reaction. B. In each DIAm fiber, the SDH reaction was linear (across the 10-min period (r²>0.99) and the maximum veolicty of the SDH reaction (SDH_max) was calculated using the Beer-Lamber-Bouger law: $d\left[NB{T}_{dfz}\right]\left(\left[\mathit{\text{fumarate}}\right]\right)=\frac{\text{d}OD}{kl}$. Where the average OD within a DIAm fiber measured every 15 s is divided by the molar extinction coefficient of NBT_dfz (k; 26,478 mol/cm) times the path length of light absorbance (l; 6 μm in this study). C. In type identified DIAm fibers, the SDH reaction (change in OD with time) was measured after addition of 1 mM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), which disrupts the proton gradient uncoupling O₂ consumption from the electron transport chain (ETC). Note that FCCP had no effect on SDH_max in any fiber type. Treatment with 1 mM rotenone to inibit complex I of the ETC and 1 mM antimycin A inhibit complex III of the ETC markedly inhibited the SDH reaction in all DIAm fiber types (p<0.0001; Bonferroni post hoc test) providing a measure of basal [NBT_dfz] accumulation. In respirometry, changes in O₂ consumption rate after addition of FCCP, rotenone and antimycin A provide a measure of maximum mitochondrial respiratory capcity. The SDH_max (in untreated and after FCCP treatment) varied across DIAm fiber types (p<0.0001; two-way ANOVA). The SDH_max of type I and IIa DIAm fibers were comparable but both were significantly higher than the SDH_max of type IIx/IIb fibers (p<0.0001; Bonferroni post hoc test). For each comparison n=15 fibers per type per animal (6 animals – 3 females and 3 males). * denotes p< 0.05.